The increasing popularity of disposable or single use bioreactors for upstream processing has been recently noted in several studies, and can be understood by considering a typical biotech manufacturing facility. The infrastructure required to implement a facility using traditional glass/steel bioreactors is substantial, and the time and expense required to construct it can be immense. The requirement that both the bioreactor itself, and also the ingress and egress tubing, utilize inert materials such as 316L electro-polished stainless steel requires a large initial investment. Additionally, the size and form factor of disposable bioreactor vessels generally lend themselves to easier storage and re-configurability when compared with traditional, rigid glass/steel solutions. Other advantages are the cost and time savings over traditional designs, the reduction in preparation and sterilization time, the reduced need for purified water for cleaning the vessel after a run, and the significantly reduced post run maintenance time. Additionally, single use bioreactors and the associated plastic tubing lend themselves to being re-configured and validated quickly and efficiently as manufacturing or process requirements change. Although a number of different styles of single use bioreactors have been conceived and introduced into the marketplace, two types currently predominate.
The first type of single-use bioreactor is generally referred to as the “pillow” or “rocker” bag style, and is described, for example, in U.S. Pat. No. 6,190,913. This style of bag has been constructed from a variety of different polymeric materials, but low density polyethylene and ethylene/vinyl-acetate copolymers are currently the most popular materials for at least the innermost layer which contacts the aqueous growth medium. This type of disposable bioreactor utilizes a wave motion induced by movement about a single axis to both mix and sparge (aerate) the contents of the bioreactor.
Another style of bioreactor bag is often referred to as a “liner style” and substantially mimics the function and form of a traditional glass/steel bioreactor. A disposable polymeric bag is used as a liner inside a generally cylindrical glass/steel tank and generally uses an impeller to mix the contents of the bioreactor vessel. This type of system has been commercialized by several manufacturers (see e.g., Published US Patent Applications 2005/0272146 and 2005/0239199). The disposable liner type of bioreactor bag has proven popular for process development or pilot runs using growth medium volumes of 25 liters or more.
Both styles of disposable bioreactors have undergone some scrutiny in order to judge their efficacy in comparison to traditional glass/steel bioreactors. However, statistically rigorous analyses are apparently not available to date. Irrespective of the style of the disposable bioreactor, the inner surface of the polymeric bioreactor bag needs to be both biologically inert and also not prone to leaching nutrients from the growth medium or from the polymer into the growth medium (see Kadarusman et. al, Growing Cholestero-Dependent NSO Myeloma Cell Line in the Wave Bioreactor System: Overcoming Cholesterol-Polymer Interaction by using Pretreated Polymer or Inert Fluorinated Ethylene Propylene, Biotechology Progress, 2005, 21, p. 1341). Additionally, the liner needs to be chemically stable under the optical illumination often used to facilitate cell growth.
Whether a bioreactor is of traditional design or a modern disposable format, the basic function of a bioreactor is to provide a controlled environment in order to achieve optimal growth/product formation in the cell or microbe that is present in the aqueous bioreactor growth medium. The traditional glass and steel bioreactors used in batch processes were proven to be effective in the course of antibiotic production in the 1950's, and still remain the dominant paradigm in the fermentation industry. Criteria for optimizing bioreactor design have been enumerated before (see A. Margaritas and J. B. Wallace, Novel Bioreactor Systems and Their Applications, Nature Bio/Technology, May 1984, p. 447.) According to Margaritas et al, some of the basic criteria required to characterize and understand bioreactor performance are:    1. Mass and heat transfer and hydrodynamic characteristics of the bioreactor,    2. Potential for bioreactor scale-up, and    3. Capital and operating costs of the bioreactor
Given the extensive existing knowledge regarding the design and performance of traditional glass/steel bioreactors, it was considered desirable to mimic their design as much as possible in the implementation of a disposable bioreactor system. Examination of current products reveals that this approach has been utilized, but with only mixed results. An example of this prior art style of product, as shown in FIGS. 1a and 1b, employs a liner made of a bio-compatible, flexible polymeric material set inside a vessel which provides structural support. The end result is a disposable liner which assumes a physical form very similar to that of a traditional (generally cylindrical) bioreactor. The use of an impeller and sparger completes the analogy to a traditional glass/steel vessel. Therefore, the performance of a liner type disposable bioreactor can be measured against the list of criteria 1 through 3 above, and one can get a reasonable idea of the performance through comparison to the performance of similar size and shape traditional glass/steel systems. Given the analogy to known bioreactor shapes and sizes, impeller design and placement, the mixing and oxygenation rates can be approximated. Additionally, traditional scaling arguments can be formulated and implemented, and known techniques of computational fluid mechanics can be applied.
The “pillow” bag style of disposable bioreactor also utilizes bags made of biocompatible films. An example of this design is shown in FIG. 2 (see V. Singh, Disposable Bioreactor for cell culture using wave-induced agitation, Cytotechnology 30, 1999, p. 149.). Most proponents of the “pillow” bag stipulate that traditional bioreactors and single-use bioreactors that emulate the traditional glass/steel bioreactor using a disposable liner limit the yield obtainable in mammalian cell growth runs due to impeller induced sheer stress of the cells during mixing and sparging. The “pillow” bag style of disposable bioreactor bag has no impeller, and utilizes a rocking motion to mix and sparge the micro-organisms contained within the bag. It is claimed that this rocking motion is gentle on the cells and does not cause sheer stress which can easily damage mammalian cells (G. Kretzmer and K. Schugerl, Journal of Applied Microbiology and Biotechnology, Vol. 34, No. 5, 613-616 (1991)). The boundary conditions set forth by the bag, combined with the rocking motion are alleged to adequately mix and aerate the aqueous culture medium when mammalian cells are used. However, it is not entirely clear that this is the case or that this style of bioreactor is efficacious for non-mammalian cell processes, such as bacterial or microbial fermentations that require more oxygen and/or can have highly viscous contents (reaction medium).
As shown in FIG. 2, the most common commercial versions of the pillow bag style single-use bioreactor utilizes one or more ports on the top of the bag to bring in the air or oxygen, and then utilizes another (output) port in relatively close proximity to the input port to vent the gas(es) produced by the bioprocess. It is likely that in many cases the incoming sparging gas is vented too quickly so that sub-optimal oxygenation occurs. For this type of bag, no evidence of an understanding of the performance criteria 1 through 3, as set forth above, is found in the literature. Published data generally indicates that the total cell density achieved in this style of bioreactor is generally less than (and at best, equal to) that achieved in small shaker flasks. It is an object of the present invention to provide a design for a single-use bioreactor vessel which ensures adequate mixing of the ingredients in the growth medium, and sufficient aeration for cells or microbes which require significant oxygen, but at the same time does not cause destructive shear stress and is scalable to comparatively large volume runs.